Bottom Line:
The viability of the cells improved following treatment with SS31 between 100 nM and 1 µM, compared with untreated control group.Compared with the t‑BHP treatment group (20.0±3.8%), the number of annexin V‑positive cells decreased dose‑dependently to 13.6±2.6, 9.8±0.5 and 7.4±2.0% in the SS‑31 treated group at concentrations of 10 nM, 100 nM and 1 µM, respectively.Treatment with SS‑31 significantly prevented the t‑BHP‑induced expression of nitrotyrosine and 8‑OHdG, decreased the quantity of mitochondrial ROS, increased mitochondrial potential, and prevented the release of cytochrome c from mitochondria into the cytoplasm.

ABSTRACTThe present study aimed to investigate the ability of SS31, a novel mitochondria‑targeted peptide to protect against t‑BHP‑induced mitochondrial dysfunction and apoptosis in 661W cell lines. The 661W cells were treated with various concentrations of SS‑31 and an MTT assay was used to determine cell viability. The expression of nitrotyrosine and 8‑hydroxydeoxyguanosine (8‑OHdG) was detected using immunofluorescent staining. Apoptosis were assessed using Hoechst staining and an annexin V/propidium iodide flow cytometer. Reactive oxygen species (ROS) were detected using MitoSOXTM with confocal microscopy. Changes in mitochondrial membrane potential were analyzed using flow cytometry. In addition, the release of cytochrome c was analyzed using confocal microscopy. The viability of the cells improved following treatment with SS31 between 100 nM and 1 µM, compared with untreated control group. Compared with the t‑BHP treatment group (20.0±3.8%), the number of annexin V‑positive cells decreased dose‑dependently to 13.6±2.6, 9.8±0.5 and 7.4±2.0% in the SS‑31 treated group at concentrations of 10 nM, 100 nM and 1 µM, respectively. Treatment with SS‑31 significantly prevented the t‑BHP‑induced expression of nitrotyrosine and 8‑OHdG, decreased the quantity of mitochondrial ROS, increased mitochondrial potential, and prevented the release of cytochrome c from mitochondria into the cytoplasm. Therefore, the SS31 mitochondria‑targeted peptide protected the 661W cells from the sustained oxidative stress induced by t‑BHP.

f3-mmr-12-04-5026: SS31 prevents apoptotic nuclei condensation and externalization of membrane phosphatidylserine residue in 661W cells. (A) Phase-contrast light micrographs revealed cells exhibiting a generally unhealthy appearance following treatment with 100 µM t-BHP for 24 h, whereas those co-cultured with 100 nM SS31 maintained a relatively healthy appearance. (magnification, ×1,000). (B) Representative images of Hoechst-labeled nuclei, condensation of chromatin and apoptotic bodies in the 661W cells following damage induced by 100 µM t-BHP for 24 h (arrows). These changes were less visible in the control cells and were markedly improved in the cells treated with 100 nM SS31. (C) Representative flow cytometric images indicated a significant decrease of annexin V-positive cells in the 661W cells treated with 100 nM SS31, compared with the cells treated with t-BHP alone for 24 h. (D) Quantitative analysis of annexin V-positive cells was measured in four independent experiments using flow cytometry. The number of annexin V-positive cells decreased in a dose-dependent manner to 13.6±2.6, 9.8±0.5 and 7.4±2.0% in following treatment with 10 nM, 100 nM and 1 µM, respectively (**P<0.001, vs. control; *P<0.001, vs. t-BHP). Data are presented as the mean ± standard error of the mean.

Mentions:
Morphologically, a significant number of 661W cells had detached from the culture dish following treatment with 100 mM t-BHP for 24 h, and those that remained exhibited cell shrinkage and shedding; whereas those co-cultured with 100 nM SS31 maintained a fairly healthy appearance. Fewer cells were observed in these cultures, partly due to cell death and partly due to cell detachment (Fig. 3A). Following Hoechst staining, the 661W cells treated with 100 mM t-BHP for 24 h exhibited substantial fragmented or condensed nuclei, characteristic of apoptotic cells, whereas few apoptotic nuclei were identified in the control 661W cells. The 661W cells, which were incubated with 100 nM SS31 had a markedly decreased number of apoptotic nuclei, compared to the t-BHP group, indicating that SS31 protected the 661W cells from oxidant-induced 661W cell death (Fig. 3B).

f3-mmr-12-04-5026: SS31 prevents apoptotic nuclei condensation and externalization of membrane phosphatidylserine residue in 661W cells. (A) Phase-contrast light micrographs revealed cells exhibiting a generally unhealthy appearance following treatment with 100 µM t-BHP for 24 h, whereas those co-cultured with 100 nM SS31 maintained a relatively healthy appearance. (magnification, ×1,000). (B) Representative images of Hoechst-labeled nuclei, condensation of chromatin and apoptotic bodies in the 661W cells following damage induced by 100 µM t-BHP for 24 h (arrows). These changes were less visible in the control cells and were markedly improved in the cells treated with 100 nM SS31. (C) Representative flow cytometric images indicated a significant decrease of annexin V-positive cells in the 661W cells treated with 100 nM SS31, compared with the cells treated with t-BHP alone for 24 h. (D) Quantitative analysis of annexin V-positive cells was measured in four independent experiments using flow cytometry. The number of annexin V-positive cells decreased in a dose-dependent manner to 13.6±2.6, 9.8±0.5 and 7.4±2.0% in following treatment with 10 nM, 100 nM and 1 µM, respectively (**P<0.001, vs. control; *P<0.001, vs. t-BHP). Data are presented as the mean ± standard error of the mean.

Mentions:
Morphologically, a significant number of 661W cells had detached from the culture dish following treatment with 100 mM t-BHP for 24 h, and those that remained exhibited cell shrinkage and shedding; whereas those co-cultured with 100 nM SS31 maintained a fairly healthy appearance. Fewer cells were observed in these cultures, partly due to cell death and partly due to cell detachment (Fig. 3A). Following Hoechst staining, the 661W cells treated with 100 mM t-BHP for 24 h exhibited substantial fragmented or condensed nuclei, characteristic of apoptotic cells, whereas few apoptotic nuclei were identified in the control 661W cells. The 661W cells, which were incubated with 100 nM SS31 had a markedly decreased number of apoptotic nuclei, compared to the t-BHP group, indicating that SS31 protected the 661W cells from oxidant-induced 661W cell death (Fig. 3B).

Bottom Line:
The viability of the cells improved following treatment with SS31 between 100 nM and 1 µM, compared with untreated control group.Compared with the t‑BHP treatment group (20.0±3.8%), the number of annexin V‑positive cells decreased dose‑dependently to 13.6±2.6, 9.8±0.5 and 7.4±2.0% in the SS‑31 treated group at concentrations of 10 nM, 100 nM and 1 µM, respectively.Treatment with SS‑31 significantly prevented the t‑BHP‑induced expression of nitrotyrosine and 8‑OHdG, decreased the quantity of mitochondrial ROS, increased mitochondrial potential, and prevented the release of cytochrome c from mitochondria into the cytoplasm.

ABSTRACTThe present study aimed to investigate the ability of SS31, a novel mitochondria‑targeted peptide to protect against t‑BHP‑induced mitochondrial dysfunction and apoptosis in 661W cell lines. The 661W cells were treated with various concentrations of SS‑31 and an MTT assay was used to determine cell viability. The expression of nitrotyrosine and 8‑hydroxydeoxyguanosine (8‑OHdG) was detected using immunofluorescent staining. Apoptosis were assessed using Hoechst staining and an annexin V/propidium iodide flow cytometer. Reactive oxygen species (ROS) were detected using MitoSOXTM with confocal microscopy. Changes in mitochondrial membrane potential were analyzed using flow cytometry. In addition, the release of cytochrome c was analyzed using confocal microscopy. The viability of the cells improved following treatment with SS31 between 100 nM and 1 µM, compared with untreated control group. Compared with the t‑BHP treatment group (20.0±3.8%), the number of annexin V‑positive cells decreased dose‑dependently to 13.6±2.6, 9.8±0.5 and 7.4±2.0% in the SS‑31 treated group at concentrations of 10 nM, 100 nM and 1 µM, respectively. Treatment with SS‑31 significantly prevented the t‑BHP‑induced expression of nitrotyrosine and 8‑OHdG, decreased the quantity of mitochondrial ROS, increased mitochondrial potential, and prevented the release of cytochrome c from mitochondria into the cytoplasm. Therefore, the SS31 mitochondria‑targeted peptide protected the 661W cells from the sustained oxidative stress induced by t‑BHP.